Abstract. Studies have demonstrated that replication stress is widespread in a variety of cancers; therefore, its exploitation for the development of therapies is an exciting area to explore. Under replication stress, cells tend to accumulate replication intermediates, often failing to complete DNA replication. In the worst-case scenarios, such replication intermediates interfere with proper segregation of chromosomes during mitosis, leading to cell death. However, accumulating evidence indicates that cancer cells appear to develop ways to override the intrinsic replication stress they harbor, sustaining their proliferation. One such way is to efficiently perform mitotic DNA synthesis, a recently discovered mechanism that operates especially under replication stress. The use of mitotic DNA synthesis allows cells to have a ?last minute? chance to resolve replication intermediates just before chromosomes segregate. Our preliminary data suggest that cancer cells perform mitotic DNA synthesis in a different mechanism than normal cells to efficiently resolve replication intermediates. Therefore, our goal of this application is to determine if the exploitation of the differential regulation of mitotic DNA synthesis impairs the proliferation of cancer cells but not that of normal cells. We also have evidence that mitotic DNA synthesis operates more frequently at certain chromosome loci, where the completion of DNA replication is intrinsically difficult and strongly inhibited under replication stress. Such loci include common fragile sites as well as telomeres. Therefore, we hypothesize that inhibition of mitotic DNA synthesis selectively in cancer cells leads to incomplete replication of these loci under replication stress, substantially impairing their proliferation. Intriguingly, there are multiple similarities between mitotic DNA synthesis and the phenomenon called alternative lengthening of telomeres (ALT), which operates in 10- 15% of cancers. Given these findings, we also postulate that inhibition of mitotic DNA synthesis can be more effective in ALT cancer cells, as it is expected to strongly impair telomere maintenance in these cells. To block mitotic DNA synthesis in cancer cells, we will inhibit RAD52, as it acts as a major promoter of this process. We will test the efficacy of RAD52 inhibition in a sensitized condition, exploiting another mechanism that cancer cells use to overcome replication stress, their over-reliance on origin licensing. As a previous studies provided evidence that partial inhibition of origin licensing induces mitotic DNA synthesis preferentially in cancer cells particularly upon depletion of ORC, a protein complex that initiates origin licensing. To test our working hypotheses, we propose the following specific aims: 1) Test if inhibition of RAD52 selectively kills ORC-depleted cancer cells, and 2) Test if inhibition of mitotic DNA synthesis impairs telomere maintenance in ALT cancers. The expected outcomes from this proposal will reveal the co-inhibition of origin licensing and mitotic DNA synthesis as a promising method for the selective killing of cancer cells.